The polybasic region of Rac1 modulates bacterial uptake independently of self-association and membrane targeting.
The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase member, is required for Rac1 self-association, membrane localization, nuclear translocation, and interaction with downstream effectors. We previously demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the effectors that requires the polybasic region for interaction, is necessary for efficient invasin-promoted uptake of Yersinia pseudotuberculosis by nonphagocytic cells. Here we further examined the role of this region in invasin-promoted uptake. Using fluorescence resonance energy transfer experiments (FRET), we determined that engagement of integrin receptors by invasin caused elevated levels of Rac1 self-association at the site of bacterial adhesion in a PBR-dependent fashion. Self-association could be disrupted using several strategies: translocation of the Yersinia YopT prenylcysteine protease into host cells, inactivation of the Rac1 isoprenylation signal that is required for membrane localization, and elimination of the PBR. Disruption in each case impaired invasin-promoted uptake. To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail. The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association. This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.
Wong
Ka-Wing
KW
Howard Hughes Medical Institute, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Mohammadi
Sina
S
Isberg
Ralph R
RR
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D016023:Integrins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D015011:Yersinia pseudotuberculosis / Q000235:genetics / Q000378:metabolism*; D015012:Yersinia pseudotuberculosis Infections / Q000235:genetics / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D016023:Integrins; D009479:Neuropeptides; C484602:Rac1 protein, mouse; C116117:YopT protein, Yersinia; C053476:invasin, Yersinia; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D003546:Cysteine Endopeptidases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein